System for producing tissue-engineered material

ABSTRACT

A system for producing a tissue-engineered material includes a hollow member and a mechanical stimulating unit. The hollow member is adapted to be implanted in a peritoneal cavity, and is to be positioned in the peritoneal cavity in a manner that a part of the hollow member contacts an inner wall surface of the peritoneal cavity for enabling formation of a biological tissue that encapsulates the hollow member. The mechanical stimulation unit is coupled to the hollow member and configured to provide a periodic mechanical stimulus to the biological tissue by periodically causing the hollow member to expand and contract. A method for producing the aforesaid tissue-engineered material is also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a division of U.S. patent application Ser. No.13/958,899 filed on Aug. 5, 2013, which claims priority of TaiwanesePatent Application No. 101140127, filed on Oct. 30, 2012.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a system and a method for producing atissue-engineered material, more particularly to a system and a methodfor producing a tissue-engineered material in a living subject.

2. Description of the Related Art

In clinical practice, many patients with atherosclerosis or chronickidney disease may encounter situations that needreplacement/transplantation of natural, artificial or tissue-engineeredblood vessels. The natural origins may be autografts,decellularize-processed allografts or xenografts, and the artificialmaterials may be made of synthetic non-biodegradable materials includingpolyester, polypropylene, and expandable-PTFE, or made of biodegradablematerials including polyglycolic acid (PGA) and poly-L-lactic acid(PLLA).

However, allografts and xenografts are rejected by the immune systems ofthe recipients, thus need thorough decellularization and otherprocesses, resulting in increased risks of the exposure to foreignproteins and process-related chemicals. Meanwhile, the artificialmaterials often cause foreign body related chronic inflammation and needfurther treatments and processes to achieve a long-term patency.

In recent years, with the breakthroughs in the tissue-engineering field,many researchers have started investigating the methods for growingautologous cells/tissues outside the human body (in vitro) that arecapable of being transplanted directly to the patients. It has beenacknowledged that mechanical properties of the tissue cultured in vitrocan be dramatically enhanced by applying a periodic mechanicalstimulation to it. However, the in vitro cultured tissues are not alwaysdurable enough to meet the mechanical strength requirements; bioreactorsthat are used to grow the in vitro autologous tissues cannot completelymimic the environment in vivo, and additives (such as bovine serum)during the culture session might cause adverse consequences.

SUMMARY OF THE INVENTION

Therefore, the object of the present invention is to provide a systemand a method for producing a tissue-engineered material in vivo.

According to one aspect of the present invention, a system for producinga tissue-engineered material in a peritoneal cavity of a living subjectincludes:

a hollow member adapted to be implanted in the peritoneal cavity,wherein the hollow member is made of a biocompatible andfluid-impermeable material that is expansible and contractible and is tobe positioned in the peritoneal cavity in a manner that a part of thehollow member contacts an inner wall surface of the peritoneal cavityfor enabling formation of a biological tissue that encapsulates thehollow member; and

a mechanical stimulation unit coupled to the hollow member andconfigured to provide, during a stimulation session, a periodicmechanical stimulus to the biological tissue being formed on the hollowmember by periodically introducing a fluid to flow into and out of thehollow member to cause the hollow member to expand and contractaccordingly.

According to another aspect of the present invention, a method forproducing a tissue-engineered material in a peritoneal cavity of aliving subject includes the following steps of:

(a) implanting a hollow member in the peritoneal cavity, wherein thehollow member is made of a biocompatible and fluid-impermeable materialthat is expansible and contractible and is positioned in the peritonealcavity in a manner that a part of the hollow member contacts an innerwall surface of the peritoneal cavity for enabling formation of abiological tissue that encapsulates the hollow member; and

(b) during a stimulation session, providing a periodic mechanicalstimulus to the biological tissue being formed on the hollow member byperiodically introducing a fluid to flow into and out of the hollowmember to cause the hollow member to expand and contract accordingly.

BRIEF DESCRIPTION OF THE DRAWINGS

Other features and advantages of the present invention will becomeapparent in the following detailed description of the preferredembodiment with reference to the accompanying drawings, of which:

FIG. 1 is a schematic diagram of a preferred embodiment of a system forproducing a tissue-engineered material in a peritoneal cavity accordingto the invention;

FIG. 2 is a schematic diagram illustrating that a hollow member of thepreferred embodiment is implanted in a peritoneal cavity; and

FIG. 3 is a flow chart of the preferred embodiment of a method forproducing a tissue-engineered material in a peritoneal cavity accordingto the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Referring to FIGS. 1 and 2, the preferred embodiment of a system forproducing a tissue-engineered material in a peritoneal cavity 901 of aliving subject includes a hollow member 3, a mechanical stimulation unit4, and a control unit 5.

The hollow member 3 is made of a biocompatible and fluid-impermeablematerial and has an elastic segment 31 that has opposite ends, that iselastically deformable (e.g., expansible and contractible), and that isto be disposed in the peritoneal cavity 901, and a pair of securingsubcutaneous segments 32 that extend from and are in fluid communicationwith the opposite ends of the elastic segment 31 respectively, that areto be extended outwardly of the peritoneal cavity 901, and that areadapted to position the elastic segment 31 in the peritoneal cavity 901in a manner that a part of the elastic segment 31 contacts an inner wallsurface of the peritoneal cavity 901 for enabling formation of abiological tissue that encapsulates the elastic segment 31.

In this embodiment, the hollow member 3 is configured in a tubularshape. However, the size and the shape of the hollow member 3 aredependent on the size and shape requirements of the tissue-engineeredmaterial and are not limited hereto. For example, the elastic segment 31of the hollow member 3 may be configured in a sac shape. Moreover, thehollow member 3 could include a plurality of elastic segments 31 withthe same or different sizes.

The mechanical stimulation unit 4 includes a pair of delivery pipe units41 each of which is coupled to and in fluid communication with arespective one of the securing segments 32, a pressurizing device 42that is coupled to and in fluid communication with one of the deliverypipe units 41 and that is operable to introduce a fluid that flows intoand out of the hollow member 3, and a pressure sensing device 43 that isconnected to the other one of the delivery pipe units 41 and that isoperable for detecting the pressure of the fluid in the hollow member 3and for generating an output signal corresponding to the pressure. Eachof the delivery pipe units 41 includes a duct member 411 that isconnected to and in fluid communication with the respective one of thesecuring segments 32, and a pipe member 412 connected to and in fluidcommunication with the duct member 411 and connected to a correspondingone of the pressurizing device 42 and the pressure sensing device 43. Inthis embodiment, the pressurizing device 42 is a syringe pump (V3syringe pump, commercially available from Kloehn, USA), but it is notlimited hereto.

The control unit 5 is electrically coupled to the pressurizing device 42and the pressure sensing device 43, and is operable for receiving theoutput signal from the pressure sensing device 43 and for controllingoperation of the pressurizing device 42 according to the output signalto control e.g., duration of the stimulation session, frequency ofintroducing the fluid, and the pressure of the fluid being introducedinto the hollow member 3. Preferably, the control unit 5 is aprogrammable device.

Referring to FIG. 3 as well as FIGS. 1 and 2, the preferred embodimentof a method according to the present invention includes the followingsteps of:

(a) implanting the hollow member 3 in the peritoneal cavity 901, whereinthe hollow member 3 is positioned in the peritoneal cavity 901 in amanner that a part of the hollow member 3 contacts an inner wall surfaceof the peritoneal cavity 901 for enabling formation of a biologicaltissue that encapsulates the hollow member 3; and

(b) during a stimulation session, providing from the mechanicalstimulation unit 4 a periodic mechanical stimulus to the biologicaltissue being formed on the hollow member 3 by periodically introducing afluid to flow into and out of the hollow member 3 to cause the hollowmember 3 to expand and contract accordingly.

Preferably, the method further includes a step:

(c) removing the hollow member 3 from the peritoneal cavity 901 andharvesting the biological tissue from the hollow member 3 to obtain thetissue-engineered material.

To be specific, in step (a), the elastic segment 31 of the hollow member3 is disposed in the peritoneal cavity 901, and the pair of securingsegments 32 extend outwardly of the peritoneal cavity 901 and areembedded in a subcutaneous space so as to position the elastic segment31 in the peritoneal cavity 901.

Preferably, in step (b), the fluid is periodically introduced into anddrawn out of the hollow member 3 from one of the securing segments 32during the stimulation session.

In step (b), duration of the stimulation session, frequency ofintroducing the fluid, and pressure of the fluid being introduced intothe hollow member 3 are adjusted by the control unit 5 throughcontrolling operation of the pressurizing device 42 according to theoutput signal from the pressure sensing device 43.

Preferably, step (b) is repeated to conduct multiple stimulationsessions within a predetermined time period.

In this embodiment, the fluid is a liquid. However, the fluid may be gaswhile implementing other embodiments according to the present invention.Also, the duration of the stimulation session, frequency of introducingthe fluid, and pressure of the fluid being introduced into the hollowmember 3 are dependent upon the size, the shape, and the material of thehollow member 3, and are not limited hereto.

EXAMPLES Example 1

For producing the tissue-engineered material of Example 1 according tothe preferred embodiment of the present invention, a silicone tube(i.e., a hollow member 3) was implanted into a peritoneal cavity 901 ofa Sprague-Dawley (SD) rat. The silicone tube has an inner diameter of 1mm, an outer diameter of 3 mm, and a length of 6 cm inside peritonealcavity, and 27 SD rats were subjected to the implantation. Whileimplanting the silicone tube, a small incision was made in each of leftand right sides of an abdominal wall of the SD rat. The silicone tubewas inserted into the peritoneal cavity 901 from one of the incisionsand had an elastic segment 31 retained in the peritoneal cavity 901. Theelastic segment 31 of the silicone tube had a part that contacts aninner surface of the peritoneum for enabling the formation of abiological tissue that encapsulates the silicone tube. The silicone tubehad a pair of securing segments 32 and each of the securing segments 32was extended outwardly from the elastic segment 31 and penetrated theabdominal wall to be sutured and embedded in subcutaneous tissues of theback of the SD rat for securing the silicone tube. Then, each of thesecuring segments 32 was coupled to and in fluid communication with aduct member 411 of a delivery pipe unit 41, wherein each of the ductmember 411 was partially inserted into the SD rat body with one endlocated outside of the SD rat body for coupling with a pipe member 412of a corresponding one of the delivery pipe units 41. The two incisionswere closed with sutures after the implantation and 7 days of a recoveryperiod was given to the SD rat before mechanical stimulation performed.

It is worth noting that the design of the silicone tube to have thesecuring segments 32 secured and embedded subcutaneously in the SD ratnot only positions the elastic segment 31 in the peritoneal cavity 901,but also prevents the germs invading the peritoneal cavity 901 from thebody surface of the SD rat via the silicone tube, so as to lower therisk of causing the infection of the peritoneal cavity 901.

After the resting period, each of the 27 SD rats was subjected to aperiodic stimulation session. During the periodic stimulation session,the pipe member 412 of each of the delivery pipe units 41 was coupled toand in fluid communication with the respective duct member 411. Asyringe pump (i.e., the pressurizing device 42) was then coupled to oneof the pipe members 412 for introducing water to flow into and out ofthe respective pipe member 412, and a pressure sensing device 43 wascoupled to the other one of the pipe members 412 for detecting the waterpressure flowing in the silicone tube and the delivery pipe units 41.The water was periodically introduced into and drawn out of the siliconetube from one of the securing segments 32 by the syringe pump so as tocause the silicone tube to expand and contract accordingly and togenerate pulsations thereof. The frequency of introducing the water intoand out of the silicone tube was about 0.3 Hz (3 to 4 seconds for apumping cycle), and the maximum water pressure being introduced into thesilicone tube during the pumping cycle was 4 Kg/cm². The relationshipbetween the water pressure and an (diameter) inflation rate of thesilicone tube based on the material property of the silicone tube may beillustrated by the following: 4.1 Kg/cm²=12%, 3.5 Kg/cm²=10.3%, and 3Kg/cm²=6.6%. The stimulation session lasted for 8 hours per day, and wasrepeated for 15 consecutive days. After 8 hours of the stimulationsession, the two pipe members 412 were disconnected from the respectiveduct members 411 to allow the SD rats to rest. After the 15 days ofrepeating stimulation sessions, the silicone tube was removed from eachof the SD rats and the biological tissue which encapsulates the elasticsegment 31 and the securing segments 32 was harvested, and thetissue-engineered material of Example 1 was obtained.

It should be noted that the tissue-engineered material encapsulated bothof the elastic segment 31 in the peritoneal cavity 901 and the securingsegments 32 subcutaneously. Therefore, the tissue-engineered material ofExample 1 was divided into a tubular sample 1(a), which was formed onthe elastic segment 31, and a tubular sample 1(b) which was formed onthe securing segments 32. Both of the samples 1(a) and 1(b) weresubjected to the following analysis.

Comparative Example 1

The method for producing the tissue-engineered material of ComparativeExample 1 is similar to that of Example 1. The difference between theExample 1 and Comparative Example 1 resides in that no mechanicalstimulus was provided to the biological tissue being formed on thesilicone tube of Comparative Example 1. That is, the silicone tube ofComparative Example 1 was not connected to the delivery pipe units 41but merely had the elastic segment 31′ thereof being positioned in theperitoneal cavity 901 and the two securing segments 32′ being suturedand embedded subcutaneously in the same SD rat as Example 1. Here, thesilicone tube for growing the tissue-engineered material of ComparativeExample 1 was implanted into the peritoneal cavity 901 together withthat of Example 1 but spaced apart from each other in order to form thebiological tissues separately. After the 15 days during which nomechanical stimulation was provided, the silicone tube of ComparativeExample 1 was removed from the peritoneal cavity 901 together with thatof Example 1, and the biological tissue of Comparative Example 1 washarvested from the respective silicone tube to obtain thetissue-engineered material of Comparative Example 1. Likewise, thetissue-engineered material of Comparative Example 1 was divided into atubular sample 2(a), which was formed on the elastic segment 31′, and atubular sample 2 (b) which was formed on the securing segments 32′. Bothof the samples 2(a) and 2(b) were subjected to the following analysis.

[Mechanical Property Analysis] 1. Compliance Testing

The tissue-engineered material of each of the tubular samples 1(a),1(b), 2(a), and 2(b) was immersed in a saline solution with two endsconnected to and in fluid communication with a mechanical testingplatform for providing a tensile stress thereto with an elongation rateof about 10%. During a testing cycle, a fluid was introduced by themechanical testing platform to flow into and out of each of the sampleswith a fluid injection (or drawing) rate of 0.2 ml/min. The fluidpressure during the testing cycle ranged from 10 mmHg to 140 mmHg, andthe total number of the testing cycle was 14. The compliance of each ofthe samples was calculated by applying the following formula (I):

$\begin{matrix}{{{Compliance}\mspace{11mu} \left( {\% \text{/}100\mspace{14mu} {mm}\; {Hg}} \right)} = {\frac{\left( {D_{140} - D_{70}} \right)/D_{70}}{70\mspace{14mu} {mm}\; {Hg}} \times 10^{4}}} & (I)\end{matrix}$

wherein D₁₄₀ represents the outer diameter of the tissue-engineeredmaterial while the fluid pressure in the samples is 140 mmHg, D₇₀representing the outer diameter of the tissue-engineered material whilethe fluid pressure in the samples is 70 mmHg. The compliance result ofeach of the samples is listed in Table 1.2. Burst pressure testing

After the compliance test, the tissue-engineered material of each of thesamples was subjected to a burst pressure testing by continuouslyintroducing the fluid into the tissue-engineered material with a feedingrate of 0.2 ml/min until the tissue-engineered material burst out andthe burst pressure of the tissue-engineered material of each of thesamples was recorded and listed in Table 1.

3. Suture Retention Force Testing

The tissue-engineered material of each of the samples was subjected to asuture retention force testing. A 6-0 Prolene suture was placed toconnect the sample to a step motor-operated extending device. The sutureneedle was placed into each of the sample edge with a 2 mm bite at oneend while the other end of each of the samples was secured, followed bydriving the step motor to increase the tension force of the suture untilthe breakage of the sample edge. The result of the maximum sutureretention force of each of the samples is listed in Table 1.

[Tissue Slicing and Colorimetric Analysis]

The tissue-engineered material of each of the samples was fixed by 10%buffered formaldehyde overnight and followed by being embedded inparaffin. Each of the samples was sectioned at 4 μm thickness and wassubjected to hematoxylin-eosin stain and Masson trichrome stain. Foreach of the samples, 4 color images were acquired with a photomicroscopeand a digital camera, and the content of collagen of each of theexamples was determined by image analysis. The result of each of thesamples is listed in Table 1.

TABLE 1 Sample Sample Sample Sample 1(a) 1(b) 2(a) 2(b) Burst 28.08 ±22.07 24.41 ± 17.34 9.39 ± 6.86 17.93 ± 11.04 Pressure (psi) mean ± SDSuture Retention 81.86 ± 49.65 82.19 ± 46.77 35.3 ± 18.28 64.75 ± 21.09Force (gw) mean ± SD Collagen Content 0.4744 ± 0.1911 0.4375 ± 0.09440.3269 ± 0.0370 0.3369 ± 0.1210 Ratio mean ± SD Compliance 1.69 ± 0.831.57 ± 0.70 2.28 ± 0.59 1.34 ± 0.36 (% per 100 mmHg) mean ± SD WallThickness (mm) 0.2349 ± 0.1546 0.1739 ± 0.1082 0.1568 ± 0.1354 0.0957 ±0.0826 mean ± SD

[Results]

As shown in Table 1, the burst pressure of Sample 1(a) is 28.08±22 0.07psi, which is obviously larger than that of Sample 2(a) (9.39±6.86 psi).The suture retention force of Sample 1(a) is 81.86±49.65 g, which isalso larger than that of Sample 2(a). In the compliance test, Sample2(a) has greater compliance result of 2.28±0.59% per 100 mmHg than thatof Sample 1(a) (1.69±0.83% per 100 mmHg). As for the tubular wallthickness, Sample 1(a) (0.2349±0.1546 mm) is thicker than Sample 2(a)(0.1568±0.1354 mm). As for the content ratio of the collagen, Sample 1(a) is higher than Sample 2(a). In general, Sample 1(a) has relativelyhigh mechanical property than that of Sample 2(a), as well as thecontent of the collagen in the tubular samples.

As for the Samples 1(b) and 2(b), the burst pressure of Sample 1(b) is24.41±17.34 psi, which is obviously larger than that of Sample 2(b)(17.93±11.04 psi). The suture retention force of Sample 1(b) is82.19±46.77 g, which is also larger than that of Sample 2(b)(64.75±21.09 g). In the compliance test, Sample 1(b) has bettercompliance result of 1.57±0.70% per 100 mmHg than that of Sample 2(b)(1.34±0.36% per 100 mmHg). As for the tubular wall thickness, Sample1(b) (0.1739±0.1082 mm) is thicker than Sample 2(b) (0.0957±0.0826 mm).As for the content of the collagen, Sample 1(b) is higher than Sample2(b).

It is worth noting that the standard deviation of the tubular wallthickness of Samples 2(a) and 2(b) are close to the corresponding meanvalues of the tubular wall thickness, illustrating that the variedtubular wall thickness occurs in the samples without the appliedmechanical stimulation.

To sum up, by using the hollow member 3 to implant in the peritonealcavity 901 of the living subject and to provide a mechanical stimulus tothe hollow member 3, the tissue-engineered material of the presentinvention is capable of being formed on the hollow member 3 and havinghigh mechanical strength. Further, since the tissue-engineered materialis formed in the living subject, it is capable of being transplanted asan autograft or other autologous transplantation which dramaticallylowers the inflammation and the rejection rate.

While the present invention has been described in connection with whatis considered the most practical and preferred embodiment, it isunderstood that this invention is not limited to the disclosedembodiment but is intended to cover various arrangements included withinthe spirit and scope of the broadest interpretation so as to encompassall such modifications and equivalent arrangements.

What is claimed is:
 1. A system for producing a tissue-engineeredmaterial in a peritoneal cavity of a living subject, said systemcomprising: a hollow member adapted to be implanted in the peritonealcavity, wherein said hollow member is made of a biocompatible andfluid-impermeable material that is expansible and contractible and is tobe positioned in the peritoneal cavity in a manner that a part of saidhollow member contacts an inner wall surface of the peritoneal cavityfor enabling formation of a biological tissue that encapsulates saidhollow member; and a mechanical stimulation unit coupled to said hollowmember and configured to provide, during a stimulation session, aperiodic mechanical stimulus to the biological tissue being formed onsaid hollow member by periodically introducing a fluid to flow into andout of said hollow member to cause said hollow member to expand andcontract accordingly.
 2. The system as claimed in claim 1, wherein saidhollow member has an elastic segment that has opposite ends, that iselastically deformable, and that is to be disposed in the peritonealcavity, and a pair of securing subcutaneous segments that extend fromand are in fluid communication with said opposite ends of said elasticsegment respectively, that are to be extended outwardly of theperitoneal cavity, and that are adapted to position said elastic segmentin the peritoneal cavity.
 3. The system as claimed in claim 2, whereinsaid mechanical stimulation unit is configured such that the fluid isperiodically introduced into and drawn out of said hollow member fromone of said securing segments during the stimulation session.
 4. Thesystem as claimed in claim 2, wherein said mechanical stimulation unitincludes a pair of delivery pipe units each of which is coupled to andin fluid communication with a respective one of said securing segments,a pressurizing device that is coupled to and in fluid communication withone of said delivery pipe units and that is operable to introduce thefluid that flows into and out of said hollow member, and a pressuresensing device that is connected to the other one of said delivery pipeunits and that is operable for detecting the pressure of the fluid insaid hollow member and for generating an output signal corresponding tothe pressure.
 5. The system as claimed in claim 4, wherein saidpressurizing device includes a syringe pump.
 6. The system as claimed inclaim 4, further comprising a control unit that is electrically coupledto said pressurizing device and said pressure sensing device, and thatis operable for receiving the output signal from said pressure sensingdevice and for controlling operation of said pressurizing deviceaccording to the output signal.
 7. The system as claimed in claim 6,wherein said control unit is configured to control operation of saidpressurizing device for controlling duration of the stimulation session,frequency of introducing the fluid, and the pressure of the fluid beingintroduced into said hollow member.
 8. The system as claimed in claim 7,wherein said control unit is a programmable device.
 9. The system asclaimed in claim 4, wherein each of said delivery pipe units includes aduct member that is connected to and in fluid communication with therespective one of said securing segments, and a pipe member connected toand in fluid communication with said duct member and connected to acorresponding one of said pressurizing device and said pressure sensingdevice.